Endotoxin is a lipopolysaccharide present in a cell wall of a Gram-negative bacterium and is the most typical pyrogen. If a transfusion, a medicine for injection, or blood contaminated with endotoxin is introduced into the human body, endotoxin may induce severe side effects such as fever and shock. Therefore, it has been required that the above-mentioned medicine or instrument is kept not to be contaminated with endotoxin.
A limulus amoebocyte lysate (hereinafter, also referred to as “LAL”) contains serine proteases which are activated by endotoxin. In the case where LAL reacts with endotoxin, a coagulogen present in LAL is hydrolyzed into a coagulin by an enzyme cascade of the serine proteases activated depending on the amount of endotoxin, and the coagulin is associated with one another to form an insoluble gel.
Examples of a method of measuring the concentration of endotoxin include a semi-quantitative gelation method which includes: leaving a sample blended with LAL to stand; turning the container upside down after a lapse of a predetermined time; and judging whether the sample gets gelation or not based on the presence or absence of dropping of the sample to examine whether or not the sample contains endotoxin above a certain concentration. Examples of another sensitive method of quantifying endotoxin include: a turbidimetric method which analyze a sample with time by determining the turbidity of a sample caused by gel formation by a reaction between LAL and endotoxin; and a colorimetric method which is performed by using a synthetic substrate which is hydrolyzed by an enzyme cascade to develop a color.
Of the above-mentioned quantification methods, the turbidimetric method may require a very long reaction time until gel formation is detected because the amount of an enzyme activated by an action of a low concentration of endotoxin is small. On the other hand, the colorimetric method has a disadvantage that the method is easily affected by the turbidity (such as blood lipid) of a sample and a pigment mixed (such as hemoglobin caused by hemolysis). Therefore, the above-mentioned turbidimetric method and colorimetric method are not necessarily the best methods for measurement of endotoxin in blood of a patient in emergency or for measurement of endotoxin in a dialysis solution or blood in artificial dialysis.
Moreover, it has been proposed a laser light scattering particle measurement method, which involves measuring formation of fine gel-particles at an aggregation early stage by: forming fine gel-particles at an early stage by stirring a sample obtained by blending LAL and endotoxin; and analyzing the sizes and number of the fine gel-particles based on the intensity of laser light scattered by the fine gel-particles. It has been reported that the method can shorten the measurement time to about one-third compared with the turbidimetric method and can reduce the effects of the turbidity or color of the sample.
However, even if the above-mentioned laser light scattering particle measurement method is used, it takes a time of 40 to 50 minutes to measure a low concentration (about 1 EU (endotoxin unit)/L) of endotoxin. In the laser light scattering particle measurement method, the optical system is complex because particles in a minute space are observed, resulting in a disadvantage that measurement of many analytes is difficult.    Patent Document 1: JP 2667695 B2